Editing Peptide Conjugation to KLH (section)

== Coupling of Peptide to KLH ==

This protocol will yield enough peptide-KLH for the immunization of two rabbits with approximately five injections per rabbit (see Hint #5).

1.Weigh out 100 mg of Keyhole Limpet Hemocyanin (KLH) and dissolve the solid in 2 ml of ddH2O.

2. Sonicate and vortex to encourage the KLH to dissolve (takes approximately 4 hours), and mix the solution by continuous rotation using a "nutator" rotating platform or similar device at 4°C.

3. Once the KLH is dissolved, dialyze the KLH solution against 2 liters of 0.1 M Sodium Phosphate, pH 7.8, overnight at 4°C (see Protocol on Dialysis of Proteins). This removes any contaminating thiols or amino compounds.

4. Centrifuge 10 minutes at full speed in a microcentrifuge to remove aggregates (see Hint #6).

5. Split the dialyzed KLH solution into 2 aliquots: one for thiol- and one for amino- coupling.

C. Amino Coupling

1. For amino coupling, add 5 mg of peptide to one aliquot of KLH. Add the peptide as a solid if it is soluble in water; otherwise, add the peptide directly from a 100 mg/ml stock of peptide dissolved in DMSO. Precipitation does not matter and often happens.

2. Add Glutaraldehyde to a final concentration of 0.1% (CAUTION! see Hint #1).

3. After adding the Glutaraldehyde, check the pH of the peptide-KLH-Glutaraldehyde reaction by spotting 2 μl onto pH paper. If the pH is low, adjust to pH 7.8 using NaOH.

4. Incubate for 8 to 12 hr at 4°C, rotating gently on a nutator or equivalent.

5. Add a tiny pinch (a few milligrams) of Sodium Borohydride (NaBH4) to quench the remaining Glutaraldehyde. Make sure the sample is in a larger tube (i.e. 15 ml conical) since it tends to fizz up.

6. Incubate the sample for 8 to 12 hr at 4°C with gentle rotation. This is the "glut conjugate".

D. Thiol Coupling

1. For the thiol coupling, warm the second aliquot of KLH solution to room temperature.

2. Add 0.11 volume of IAA-NHS ester.

3. Incubate at room temperature. After 10 min, the KLH-IAA-NHS reaction will start to get a little cloudy.

4. Load the reaction onto a P-10 (Bio-Rad) column equilibrated with 0.1 M Sodium Phosphate, pH 7.8. Make sure the column is at least 10 times the volume of the sample.

5. Elute the KLH-IAA-NHS with 0.1 M Sodium Phosphate, pH 7.8.

6. Pool the KLH containing fractions by color (greyish green).

7. Add 5 mg of peptide to the KLH containing fractions. Add the peptide as a solid if it is soluble in water; otherwise add peptide directly from a 100 mg/ml stock of peptide dissolved in DMSO. Precipitation does not matter and often happens.

8. Add Glutaraldehyde to a final concentration of 0.1%.

9. After adding the Glutaraldehyde, check the pH of the peptide-KLH-glutaraldehyde reaction by spotting 2 μl onto pH paper. If the pH is low, adjust to pH 7.8 using NaOH.

10. Incubate for 8 to 12 hr at 4°C, rotating gently on a nutator or equivalent.

11. Add a tiny pinch (a few milligrams) of NaBH4 to quench the remaining Glutaraldehyde. Make sure the sample is in a larger tube (i.e. 15 ml conical) since it tends to fizz up.

12. Incubate the sample for 8 to 12 hr at 4°C with gentle rotation. This is the "glut conjugate".

13. Pool the coupled peptides (glut conjugates) from the amino and thiol couplings.

Dilute to 5 ml and add NaCl to 0.15 M final concentration. If there is a precipitate, sonicate vigorously to break it up.

14. Split the peptide-KLH into 1 ml aliquots (each aliquot will immunize two rabbits) and freeze them to store.